Cartridge, electrowetting sample processing system and droplet formation

ABSTRACT

A cartridge for use in an electrowetting sample processing system, the cartridge having at least one inlet port for introducing an input liquid in an internal gap of the cartridge, wherein the gap has at least one hydrophobic surface and is configured to provide an electrowetting induced movement of a microfluidic droplet of input liquid, wherein the input liquid has a carrier liquid and a processing liquid and the gap has a capture zone that is configured to capture at least a part of the processing liquid as a microfluidic droplet by use of electrowetting force and the gap further has a transfer zone that is configured to provide a passage for the carrier liquid next to the microfluidic droplet, while processing liquid is captured in the capture zone.

TECHNICAL FIELD OF THE INVENTION

The current invention relates to a cartridge, in particular a disposablecartridge for use in an electrowetting sample processing system anelectrowetting sample processing system and a method for operating sucha cartridge or system.

DESCRIPTION OF THE RELATED ART

Known embodiments of such cartridges are disclosed for example in WO2014/135232 A1, describing an in-cartridge separation of a droplet froma larger liquid volume previously inserted into the gap.

SUMMARY OF THE INVENTION

It is a task of the current invention to provide a cartridge that allowsfor a precise and versatile processing of microfluidic droplets.

This task is solved by a cartridge with the features of claim 1. Furtherembodiments of the cartridge, an electrowetting sample processing systemwith or without such a cartridge, as well as a method for operating sucha cartridge or system are defined by the features of further claims.

A cartridge according to the invention, in particular a disposablecartridge for use in an electrowetting sample processing system,comprises a at least one inlet port for introducing an input liquid inan internal gap of the cartridge. The gap comprises at least onehydrophobic surface and is configured to provide an electrowettinginduced movement of a microfluidic droplet of input liquid. The inputliquid comprises a carrier liquid and a processing liquid. The gapcomprises a capture zone that is configured to capture at least a partof the processing liquid as a microfluidic droplet by use ofelectrowetting force. The gap further comprises a transfer zone that isconfigured to provide a passage for the carrier liquid next to themicrofluidic droplet, while processing liquid is captured in the capturezone.

This allows an easy and precise separation of a large variety ofprocessing liquids and carrier liquids.

In an embodiment, the cartridge comprises a first part with the inletport and a second part attached to the first part, such that the gap isformed between the first part and the second part. The first part can bea top layer of the cartridge and the second part can be a bottom layerof the cartridge or vice-versa.

In an embodiment, the first part comprises a rigid body and/or thesecond part comprises or is an electrode support element or a flexiblefilm, in particular a polymer film and/or an electrically isolatingfilm.

In an embodiment, the gap is defined by a spacer that is arrangedbetween the first part and the second part and/or by the shape of atleast one of the two parts of the cartridge, in particular by a flexiblepart or a rigid part of the cartridge, and wherein in particular thesecond part is attached to a peripheral side structure of the firstpart.

In an embodiment, the cartridge comprises at least one electrode, inparticular an electrode array for applying an electrowetting force tothe microfluidic droplet. A plurality of electrodes can be arranged in afirst lateral direction and in a second lateral direction, perpendicularto the first lateral direction. The size of an electrode can be in therange of approximately 1.5×1.5 mm. The cartridge can have several zonesthat are separated from one another by at least one separation zone or aseparation wall. All zones are connected to a delivery zone. In anembodiment, the cartridge comprises an inlet channel for transferringthe processing liquid from the inlet port to the gap, wherein inparticular the inlet channel is arranged substantially perpendicular tothe orientation of the gap. Alternatively, the inlet channel is orientedat an angle of less than 90° to the orientation of the gap. For example,the inlet channel can also be oriented parallel to the orientation ofthe gap.

In an embodiment, the input liquid comprises a carrier liquid, inparticular an electrowetting filler liquid, further in particular asilicone oil.

In an embodiment, the cartridge is configured to capture the processingliquid, which comprises at least one of: a reagent liquid, a buffer, adiluent, an extraction liquid, a washing liquid and a suspension, whichfurther in particular is a suspension of magnetic beads, single cells orcell aggregates.

In an embodiment, the cartridge is configured to be operated with acarrier liquid that is an electrowetting filler liquid, further inparticular a silicone oil.

In an embodiment, the cartridge is configured to receive the inputliquid, in which the carrier liquid encloses the processing liquid,sequentially and/or alternatingly.

In an embodiment, the cartridge is configured to provide the transferzone by an open space, which is located between the inlet port and thetop of the microfluidic droplet captured in the capture zone.

In an embodiment, the transfer zone is configured to provide an axis offlow that is arranged with an offset from the center of the capturedprocessing liquid, in particular with an offset of at least a quarter ofa largest diameter of the microfluidic droplet, further in particular atleast half of the largest diameter of the microfluidic droplet.

In an embodiment, the cartridge comprises at least one capture electrodethat is located adjacent to the inlet port such that this captureelectrode covers less than 50% of the inlet port.

In a further embodiment the cartridge comprises at least one capturezone that is located closest to the inlet port such that the area of thecapture zone covers between 5% and 95% of the opening area of the inletport, in particular between 10% and 90%, further in particular between25% and 75%.

In a further embodiment the cartridge comprises at least at least onecapture electrode that is located closest to the inlet port (19′) suchthat the area of the capture electrode covers between 5% and 95% of theopening area of the inlet port (19′), in particular between 10% and 90%,further in particular between 25% and 75%.

The term “covering” describes a geometrical overlapping configuration ina projection longitudinally to an axis of the inlet port and/or along anaxis of flow exiting the opening area of the inlet port. Thiscorresponds to a visual appearance viewed along an optical axis that isperpendicular to the electrode array in a direction towards theelectrode array.

The above configurations ensure that the inlet port is not blocked bythe captured microfluidic droplet. This is achieved by activatingelectrodes to the side of the inlet port.

In an embodiment, the cartridge is configured to receive the processingliquid that comprises multiple parts, in particular parts of differentcompositions, and to accumulate these parts for providing themicrofluidic droplet.

In an embodiment, the cartridge is configured to receive at least onepart of the processing liquid that comprises a volume that isinsufficient for a transportation by electrowetting and/or thatcomprises a volume of less than 2 μl, in particular less than 1.5 μl.

In an embodiment, the cartridge is configured to capture or toaccumulate a microfluidic droplet of less than 10 μl in volume, inparticular of less than 3 μl in volume.

In an embodiment, the inlet port comprises a sealing surface for a tubeto be inserted into the inlet port. In particular, the inlet port isfunnel-shaped with an enlarged opening towards the tube to be inserted.The funnel-shape can be realized by a cone, in particular by a centeringcone.

The features of the above-mentioned embodiments of the cartridge can beused in any combination, unless they contradict each other.

An electrowetting sample processing system according to the invention,in particular a biological sample processing system comprising acartridge according to anyone of the above-mentioned embodiments.

An electrowetting sample processing system according to the inventioncomprises at least one inlet port for introducing an input liquid and aninternal gap that comprises at least one hydrophobic surface and that isconfigured to manipulate a microfluidic droplet separated from the inputliquid, if an electrowetting force is applied to the at least onemicrofluidic droplet. The input liquid comprises a processing liquid anda carrier liquid. The gap comprises a capture zone that is configured tocapture at least a part of the processing liquid by use ofelectrowetting force and the gap further comprises a transfer zone thatis configured to provide a passage for the carrier liquid from the inletport to the gap, while processing liquid is captured in the capturezone.

In an embodiment, the electrowetting sample processing system comprisesat least one electrode, in particular an electrode array, for applyingan electrowetting force to the processing liquid and/or the microfluidicdroplet.

In an embodiment, the electrowetting sample processing system comprisesa flexible cartridge, which is reversibly attachable to the electrodesof the electrowetting sample processing system, wherein in particularthe cartridge comprises a flexible second part, further in particular aflexible film or membrane.

In an embodiment, the electrowetting sample processing system or thecartridge comprises a processing zone, which is configured forprocessing samples, in particular for processing a biological sample,and/or which is operably connected to the delivery zone.

In an embodiment, the processing zone is configured for processing atleast one of:

-   -   a chemical reaction,    -   a washing process,    -   a heating process,    -   a mixing process,    -   a dilution, and    -   a hybridization.

In an embodiment, the processing zone is configured for processing a PCR(Polymerase chain reaction) process and/or a hybridization.

In an embodiment, at least one electrode comprises at least one captureelectrode that is configured to capture at least a part of theprocessing liquid as a microfluidic droplet by use of electrowettingforce. In particular, the closest edge of the capture electrode isarranged with an offset from the axis of flow of the inlet port, furtherin particular with an offset of at least a quarter or at least half of alargest diameter of the capture electrode.

In an embodiment, at least one electrode comprises at least one captureelectrode that is configured such that the area of the capture electrodecovers more than 5% of the opening of the inlet port, in particular morethan 10%, and/or less than 50% of the opening of the inlet port.

In a further embodiment, the one of the at least one capture electrodelocated closest to the inlet port covers between 5% and 95% of theopening area of the inlet port, in particular between 10% and 90%,further in particular between 25% and 75%.

Every electrode can be configured to be a capture electrode, byactivating it. That is all activated electrodes are capture electrodes.By means of a control, every electrode can be brought to an activatedstate or a non-activated state.

In an embodiment, at least one electrode of the electrowetting sampleprocessing system comprises at least one capture electrode that islocated adjacent to the inlet port such that this capture electrodecovers less than 50% of the inlet port.

In an embodiment, the at least one electrode comprises a transportelectrode for removing the microfluidic droplet from the capture zone.Every electrode can be configured as a transport electrode. Theactivated electrodes in the inlet port region are called captureelectrodes. After enough processing liquid has been accumulated by thecapture electrodes, no new processing liquid is accumulated, and thecapturing electrodes become transport electrodes. If an electrodeadjacent to the capture electrodes is activated, the capturedmicrofluidic droplet is also drawn to the newly activated electrode. Ifsubsequently, the electrode closest to the inlet port is switched off,i.e. is deactivated, the microfluidic droplet moves away from the inletport region. Thus, by activating adjacent electrodes and deactivatingelectrodes on the opposite side of the microfluidic droplet, themicrofluidic droplet can be moved in any direction within the gap.

In an embodiment, the electrowetting sample processing system comprisesa controller and/or an electrical interface for providing electricalcontrol signals to the at least one electrode.

In an embodiment, the electrowetting sample processing system comprisesa liquid feeder or liquid supply channel that is operatively connectedto the inlet port by a tube, in particular a flexible tube, for feedingan input liquid of predetermined volume to the inlet port.

In an embodiment, the liquid feeder is configured to provide the inputliquid as at least one sequential and/or alternating feed of theprocessing liquid and the carrier liquid.

In an embodiment, the electrowetting sample processing system comprisesa detector for monitoring the feed of the input liquid in particular theprocessing liquid and/or the carrier liquid. The detector can bearranged before the inlet port or after it. For example, the detectorcan be arranged at the liquid feeder or within the gap in the inlet portregion of the gap. There can also be more than one detector, for exampleone at the feeder and one in the gap.

In an embodiment, the electrowetting sample processing system comprisesa controller for operating the liquid feeder, in particular a dropletgenerator independently and/or asynchronously from the operation ofelectrodes used for electrowetting.

The features of the above-mentioned embodiments of the electrowettingsample processing system can be used in any combination, unless theycontradict each other.

A method for operating the cartridge according to the invention or foroperating the sample processing system according to the invention.

A method for operating a cartridge according to the invention thatcomprises an inlet port and an internal gap with a capture zone (62) anda transfer zone, the method comprising:

-   -   providing an input liquid that comprises a processing liquid and        a carrier liquid;    -   separating at least a part of the processing liquid in the        capture zone by use of electrowetting force;    -   transferring the carrier liquid from the inlet port to the gap        via the transfer zone, while the processing liquid is captured        in the capture zone; and    -   capturing at least a part of the processing liquid in the        capture zone for providing a microfluidic droplet that is        movable by applying an electrowetting force to the microfluidic        droplet.

In an embodiment, the step of providing the input liquid is accomplishedby sequentially and/or alternatingly feeding the processing liquid andthe carrier liquid.

In an embodiment, the input liquid comprises multiple liquid parts, inparticular parts of different compositions, and the capturing isaccomplished by accumulating these parts for providing the microfluidicdroplet.

In an embodiment, the input liquid comprises at least one part thatcomprises a volume that is insufficient for a transportation byelectrowetting and/or that comprises a volume of less than 2 μl, inparticular less than 1.5 μl.

The minimum value depends on the size of the electrodes and the gapsize, for example, a system with smaller electrodes allows to controlsmaller microfluidic droplets.

In a further embodiment, the method comprises sequentially actuatingelectrodes for inducing a motion of the microfluidic droplets away fromthe capture zone, thereby enabling a following part of the processingliquid to be captured.

The features of the above-mentioned embodiments of the method can beused in any combination, unless they contradict each other.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the current invention are described in more detail in thefollowing with reference to the figures. These are for illustrativepurposes only and are not to be construed as limiting. It shows

FIG. 1 an overview over an exemplary digital microfluidics system thatis equipped with a central control unit and a base unit, with fourcartridge accommodation sites and with four board accommodation sitesfor receiving a electrode board that each comprises an electrode array;

FIG. 2 a section view of one cartridge accommodation site with adisposable cartridge according to FIG. 1 therein; the electrode arraybeing located on a fixed bottom substrate;

FIG. 3 a section view of a further exemplary cartridge accommodationsite according to FIG. 2, wherein the electrode array is a part of thecartridge;

FIG. 4 a section view of an exemplary cartridge accommodation site witha disposable cartridge according to a further embodiment accommodatedtherein; the cartridge comprising a flexible bottom layer;

FIG. 5 a schematic view of the inlet port region of a disposablecartridge according to the invention with optional droplet detectors,wherein the inlet port being arranged at the top of the cartridge;

FIG. 6 a schematic view of the inlet port region of a disposablecartridge according to the invention, wherein the inlet port beingarranged at the side of the cartridge;

FIG. 7 a schematic view of a first embodiment of an inlet port accordingto the invention;

FIG. 8 a detailed view of FIG. 7;

FIG. 9 a schematic view of the inlet port region of FIG. 5, wherein theprocessing liquid being accumulated to a microfluidic droplet within thecartridge;

FIG. 10 a schematic depiction of several steps of the introduction of amicrofluidic droplet into the gap of the cartridge, wherein themicrofluidic droplet being separated from a larger quantity ofprocessing liquid;

FIG. 11 a schematic depiction of several steps of the introduction of amicrofluidic droplet into the gap of the cartridge, wherein themicrofluidic droplet being captured by multiple electrodes arrangedbelow the cartridge;

FIG. 12 a schematic depiction of several inlet port and captureelectrode configurations; and

FIG. 13 a schematic depiction a microfluidic droplet being captured bymultiple electrodes.

DETAILED DESCRIPTION OF THE INVENTION

The FIG. 1 shows an overview over an electrowetting sample processingsystem exemplary shown as digital microfluidics system 1 that isequipped with a central control unit 14 and a base unit 7, with fourcartridge accommodation sites 8 that each comprise an electrode array 9,and a cover plate 12. The digital microfluidics system 1 is configuredfor manipulating samples in microfluidic droplets 23, simply calledliquid droplets 23, within cartridges designed as disposable cartridges2. This digital microfluidics system 1 also comprises four boardaccommodation sites 40 for receiving an electrode board 41. The droplets23 can be a microfluidic droplet and/or a liquid comprising at least oneof a reagent, a buffer, a diluent, an extraction liquid, a washingliquid and a suspension, which in particular is a suspension of magneticbeads, single cells or cell aggregates. Samples are for example DNA(Desoxyribonucleic acid), RNA (Ribonucleic Acid), derivatives thereof,proteins, cells, or other biologically or biochemically derivedmolecules or combinations thereof.

The digital microfluidics system 1 comprises a base unit 7 with at leastone cartridge accommodation site 8 that is configured for taking up adisposable cartridge 2. The digital microfluidics system 1 can be astandalone and immobile unit, on which a number of operators are workingwith cartridges 2 that they bring along. The digital microfluidicssystem 1 thus may comprise a number of cartridge accommodation sites 8and a number of electrode arrays 9 at least some of which are located onelectrode boards 41.

It may be preferred to integrate the digital microfluidics system 1 intoa liquid handling workstation or into a Freedom EVO® roboticworkstation, so that a pipetting robot can be utilized to transferliquid portions and/or sample containing liquids to and from thecartridges 2. Alternatively, the system 1 can be can be configured as ahand-held unit which only comprises and is able to work with a lownumber, e.g. a single disposable cartridge 2. Every person of skill willunderstand that intermediate solutions that are situated in-between thetwo extremes just mentioned will also operate and work within the gistof the present invention.

According to the present invention, the digital microfluidics system 1also comprises at least one board accommodation site 40 for taking up anelectrode board 41 which comprises an electrode array 9 thatsubstantially extends in a first plane and that comprises a number ofelectrodes 10. Such an electrode board 41 preferably is located at eachone of said cartridge accommodation sites 8 of the base unit 7.Preferably each electrode array 9 is supported by a bottom substrate 11.It is noted that the expressions “electrode array”, “electrode layout”,and “printed circuit board (PCB)” are utilized herein as synonyms.

The digital microfluidics system 1 may also comprise at least one coverplate 12 with a top substrate; though providing of such cover plates 12is particularly preferred, at least some of the cover plates may bedispensed with or may be replaced by an alternative cover for holding adisposable cartridge 2 in place inside the base unit of themicrofluidics system 1. Thus, at least one cover plate 12 may be locatedat one of said cartridge accommodation sites 8. The cover plate 12 andthe bottom substrate 11 with the electrode array 9 or PCB define a spaceor cartridge accommodation site 8 respectively. In a first variant (seethe two cartridge accommodation sites 8 in the middle of the base unit7, the cartridge accommodation sites 8 are configured for receiving aslidingly inserted disposable cartridge 2 that is movable in a directionsubstantially parallel with respect to the electrode array 9 of therespective cartridge accommodating site 8. Such front- or top-loadingcan be supported by a drawing-in automatism that, following a partialinsertion of a disposable cartridge 2, transports the cartridge 2 to itsfinal destination within the cartridge accommodation site 8, where thecartridge 2 is precisely seated. Preferably, these cartridgeaccommodation sites 8 do not comprise a movable cover plate 12. Aftercarrying out all intended manipulations to the samples in liquiddroplets, the used cartridges 2 can be ejected by the drawing-inautomatism and transported to an analysis station or discarded.

In a second variant (see the two cartridge accommodation sites 8 on theright and left of the base unit 7), the cartridge accommodation sites 8comprise a cover plate 12 that is configured to be movable with respectto the electrode array 9 of the respective cartridge accommodating site8. The cover plate 12 preferably is configured to be movable about oneor more hinges 16 and/or in a direction that is substantially normal tothe electrode array 9.

Similar to the possibilities for inserting a disposable cartridge 2 intoa cartridge accommodation site 8, possibilities for inserting theelectrode board 41 into a board accommodation site 40 comprise thefollowing alternatives:

(a) vertically lowering the electrode board 41 through the respectivecartridge accommodation site 8 and into the board accommodation site 40;(b) horizontally sliding the electrode board 41 below the respectivecartridge accommodation site 8 and into the board accommodation site 40;(c) horizontally sliding the electrode board 41 below the respectivecartridge accommodation site 8 and substantially vertically lifting intothe board accommodation site 40.

In FIG. 1, there is drawn only one electrode board 41 that slidingly canbe inserted by front loading below the second cartridge accommodationsite 8 (as counted from the left).

All possible places for locating a board accommodation site 40 areindicated and pointed to by dashed arrows.

The digital microfluidics system 1 also comprises a central control unit14 for controlling the selection of the individual electrodes 10 of saidat least one electrode array 9 and for providing these electrodes 10with individual voltage pulses for manipulating liquid droplets withinsaid cartridges 2 by electrowetting. As partly indicated in FIG. 1,every electrode 10 is operatively connected to the central control unit14 and therefore can be independently or commonly addressed by thiscentral control unit 14, which also comprises the appropriate sourcesfor creating and providing the necessary electrical potentials in a wayknown in the art.

The at least one cover plate 12 preferably comprises an electricallyconductive material that extends in a second plane and substantiallyparallel to the electrode array 9 of the cartridge accommodation site 8the at least one cover plate 12 is assigned to. It is particularlypreferred that this electrically conductive material of the cover plate12 is configured to be not connected to a source of an electrical groundpotential. The cover plate 12 can be configured to be movable in anyarbitrary direction and no electrical contacts have to be taken in intoconsideration when selecting a particularly preferred movement of thecover plate 12. Thus, the cover plate 12 may be configured to be alsomovable in a direction substantially parallel to the electrode array 9and for carrying out a linear, circular or any arbitrary movement withrespect to the respective electrode array 9 of the base unit 7.

The FIG. 2 shows a section view of one exemplary cartridge accommodationsite 8 with the disposable cartridge 2 according to FIG. 1 accommodatedtherein. The disposable cartridge 2 comprises a bottom layer 3 as asecond part of the cartridge 2, a top layer 4 as a first part of thecartridge 2, and a spacer 5 that defines a gap 6 between the bottom andtop layers 3,4 for manipulating samples in liquid droplets 23 in thisgap 6.

The cover plate 12 is mechanically connected with the base unit 7 of thedigital microfluidics system 1 via a hinge 16; thus, the cover plate 12can swing open and a disposable cartridge 2 can be placed on thecartridge accommodation site 8 via top-entry loading (see FIG. 1). Anelectrically conductive material 15 of the cover plate 12 is configuredas a thin metal plate or metal foil that is attached to the topsubstrate 13. Alternatively, the electrically conductive material 15 ofthe cover plate 12 is configured as a metal layer that is deposited ontothe top substrate 13. Such deposition of the conductive material 15 maybe carried out by chemical or physical vapor deposition techniques asthey are known per se.

The cover plate 12 is configured to apply a force to a disposablecartridge 2 that is accommodated at the cartridge accommodation site 8of the base unit 7. This force urges the disposable cartridge 2 againstthe electrode array 9 in order to position the bottom layer 3 of thecartridge as close as possible to the surface of the electrode array 9.This force also urges the disposable cartridge 2 into the perfectposition on the electrode array 9 with respect to a piercing facility 18of the cover plate 12. This piercing facility 18 is configured forintroducing sample droplets into the gap 6 of the cartridge 2. Thepiercing facility 18 is configured as a through hole 19 that leadsacross the entire cover plate 12 and that enables a piercing pipette tip20 to be pushed through and pierce the top layer 4 of the cartridge 2.The piercing pipette tip 20 may be a part of a handheld pipette (notshown) or of a pipetting robot (not shown).

In the case shown in FIG. 2, the electrode array 9 is covered by adielectric layer 24. The electrode array 9 is fixed to a bottomsubstrate 11 and every individual electrode 10 is electrically andoperationally connected with the central control unit 14 (only threeconnections of the ten electrodes 10 are drawn here). The electrodearray 9 is located on an immovably fixed bottom substrate 11. Thedigital microfluidics system 1 is configured for manipulating samples inliquid droplets 23 within disposable cartridges 2 that contain a gap 6.Accordingly, the samples in liquid droplets 23 are manipulated in thegap 6 of the disposable cartridge 2. The disposable cartridge 2comprises the bottom layer 3, the top layer 4, and the spacer 5 thatdefines the gap 6 between the bottom and top layers 3,4 for manipulatingsamples in liquid droplets 23 in this gap 6. The bottom layer 3 and thetop layer 4 comprise a hydrophobic surface 17 that is exposed to the gap6 of the cartridge 2. The bottom layer 3 and the top layer 4 of thecartridge 2 are entirely hydrophobic films or at least comprise ahydrophobic surface that is exposed to the gap 6 of the cartridge 2. Thespacer 5 of the cartridge 2 may optionally be configured as a body thatincludes compartments 21 for reagents needed in an assay that is appliedto the sample droplets in the gap 6 (dotted lines).

In one example, the bottom substrate 11 or the PCB that contains theelectrode array 9 or the electrodes 10 has an electrical connector,which connects to a relay PCB, which is connected to a control PCB,wherein the control PCB is part of the central control unit 14.

FIG. 3 shows a section view of a further exemplary cartridgeaccommodation site according to FIG. 2 with a cartridge 2, wherein—incontrast to FIG. 2—the cartridge 2 comprises an electrode array 9′ ofindividual electrodes 10.

Further the cartridge 2 comprises an upper part 4, a spacer 5, ahydrophobic layer 3″, a support element 11′ for the electrode array 9′,an optional through hole 19, a liquid input port 19′ and electricallyconductive material. The upper part 4 and the spacer 5 may be providedas separate parts or in form of a single piece. The hydrophobic layer3″, the electrode array 9′ and the support element 11′ form the lowerpart of the cartridge. The electrode array 9′ is arranged between thehydrophobic layer 3″ and the support element 11′ and the gap is formedbetween the upper part 4 and the hydrophobic layer 3″. Further, thehydrophobic layer 3″ is attached to a peripheral side structure of theupper part 4 resp. to the spacer 5. The support element 11′ furthercomprises electrical connectors 14′, which are connected via multipleelectrical wires to the electrode array 9′. In turn, the electricalconnectors 14′ provide for a connection to a central control unit 14such that the electrical connectors 14′ implement an electricalinterface between cartridge 2 and the digital microfluidics system 1.The electrical interface can also be implemented by a contact field,i.e. a plurality of electrically conductive, mutually insulated contactareas.

FIG. 4 shows section view of one cartridge accommodation site 8 with adisposable cartridge 2 according to a further embodiment accommodatedtherein. Again, the electrodes 10 are arranged on and fixed to thebottom substrate 11. Again, the disposable cartridge 2 comprises abottom layer 3′ and a top layer 4. Attached to the disposable cartridgeis a spacer 5 that defines a gap 6 between the bottom and top layer 3, 4for manipulating samples in liquid droplets 23 in this gap 6. In thisembodiment, the bottom layer is a flexible bottom layer, for example amembrane 3′, for example with a hydrophobic surface 17. For example, themembrane 3′ is a 8 to 50 μm thick polypropylene film. An inlet port 19′for introducing liquid into the gap 6 is provided in the top layer 4 ofthe cartridge 2.

Preferably, the flexible bottom layer 3 is reversibly attached to theelectrodes 10 in an electrowetting sample processing system 1. Thespacer 5 may be a part of the cartridge 2 or a part of theelectrowetting sample processing system 1. In one example, the spacer 5comprises stainless steel, aluminum, hard plastic, in particular COP orceramic. The spacer 5 may be designed to define the height of the gap 6.The spacer 5 may additionally serve as a gasket for sealing the gap 6.

FIG. 5 shows a schematic view of the inlet port region of a disposablecartridge 2 according to the invention with optional droplet detectors70, 71, wherein the inlet port 19′ is arranged at the top of thecartridge 2, i.e. at the top layer 4. An input liquid 60, 61 can beintroduced into the gap 6 of the cartridge 2 by means of the inlet port19′. The input liquid comprises a carrier liquid 60 and a processingliquid 61. A quantity of processing liquid 61, surrounded by carrierliquid 60 is supplied to the inlet port 19′ by means of a supply channel50. The quantity of processing liquid corresponds to that of a liquiddroplet 23, which is the ideal amount of liquid for the furtherprocessing within the cartridge 2. The number of processing liquiddroplets within the supply channel and their sizes can be determined bymeans of a first droplet detector 70. In one example the second dropletdetector 70 monitors an optical characteristic, in particular an opticaltransmissivity and/or reflexivity, of the carrier liquid 60 and/or theprocessing liquid 61. In another example the second droplet detector 70monitors an electrical characteristic, in particular a capacitanceand/or a resistance, of the carrier liquid 60 and/or the processingliquid 61. The introduced processing liquid 61 is captured by theactivated electrodes 10′ and forming the liquid droplet 23, whereby thecarrier liquid 60 can bypass the droplet 23 on all sides and enter thefree space of the gap 6. The presence of a droplet 23 in the gap 6 ofthe cartridge 2 can additionally or alternatively be detected by meansof a second droplet detector 71.

In one example the second droplet detector 71 monitors an electricalcharacteristic between the activated electrodes 10′ and thenon-activated electrode underneath the entrance of the inlet port 19′,in particular a potential difference.

FIG. 6 shows a schematic view of the inlet port region of a disposablecartridge 2 according to the invention, wherein the inlet port 19′ isarranged at the side of the cartridge 2. In the depicted case, the inletport 19′ is arranged in the spacer 5. Of course, the droplet detectors70, 71 can also be used together with this embodiment.

FIG. 7 shows a schematic view of a first embodiment of an inlet port 19′according to the invention. A first connecting sleeve 80 is arranged atthe top of the top layer 4. The first connecting sleeve 80 is formedintegrally with the top layer 4. The first connecting sleeve 80comprises a centering cone 81 at its inside, wherein the centering cone81 faces away from the top layer 4 and widens with an increased distanceto the top layer 4. The supply channel 50 comprises a tube 51 and asecond connecting sleeve 52 with a centering cone 53 at its inside.During the assembly, the tube 51 is centered by the centering cone 81 ofthe first connecting sleeve 80. When completely inserted, the tube 51forms a tight connection with the first connecting sleeve 80. Thecentering cone 53 of the second connecting sleeve 52 faces the top layer4 and widens with a reduced distance to the top layer 4. The inside ofthe second connecting sleeve 52 is bigger than the outside of the firstconnecting sleeve 80. During the assembly, the second connecting sleeve52 is centered by the outside of the first connecting sleeve 80. Thefree inner space in the inlet port 19′ forms the inlet channel 19″.

FIG. 8 shows a detailed view of FIG. 7, wherein a liquid droplet 23 iscaptured within the capture zone 62 within the gap 6 by means of theactivated electrodes 10′ and wherein the carrier liquid 60 bypasses thedroplet 23 in the transfer zone within the gap 6.

FIG. 9 shows a schematic view of the inlet port region of FIG. 5,wherein the processing liquid 61 being accumulated to a liquid droplet23 within the cartridge 2. Small quantities of processing liquid 61,enclosed by carrier liquid 60, are introduced in the cartridge 2 throughthe inlet port 19′. The introduced processing liquid 61 accumulates inthe capture zone, i.e. in the zone of influence of the activatedelectrodes 10′. The carrier liquid 60 bypasses the capture zone. Whenthe accumulated processing liquid 61 has reached the required size ofthe liquid droplet 23, the droplet 23 is moved away from the inlet port19′ by altering the state of some of the electrodes 10, i.e. byactivating some of the neighboring electrodes 10 and by deactivatingsome of the electrodes 10 closest to the inlet port 19′.

FIG. 10 shows a schematic depiction of several steps of the introductionof a microfluidic droplet 23 into the gap 6 of the cartridge 2, whereinthe droplet 23 is separated from a larger quantity of processing liquid61. A quantity of processing liquid 61 that is bigger than the requiredamount for a liquid droplet 23 is introduced in the gap 6 of thecartridge 2 through the inlet port 19′. Again, the processing liquid 61accumulates in the capture zone, due to the activated electrodes 10′.When the desired amount of processing liquid 61 for a liquid droplet 23is accumulated, the liquid droplet 23 is sheared off the larger quantityof processing liquid 61 by activating by activating some of theneighboring electrodes 10 and by deactivating some of the electrodes 10closest to the inlet port 19′.

FIG. 11 shows a schematic depiction of several steps of introducing amicrofluidic droplet into the gap of the cartridge according to the FIG.4. Further, FIG. 4 illustrates the method according to the invention.

In this example, the figure shows a top view of the membrane 3′(transparent) with the electrode array 9 arranged underneath themembrane 3′. The electrode array 9, i.e. the set of individualelectrodes 10, is positioned such that the electrode closest to theinlet port 19′ (i.e. a capture electrode) covers less than 50% of theopening of the inlet port 19′ (indicated as circle divided in half).

In a first step to the method, the electrodes 10 are activated to becomeactivated electrodes 10′ in anticipation of the arrival of a droplet viathe inlet port 19′. The activates electrodes 10′ define the capture zone62, wherein the number of activated electrodes 10′ depends on the volumeto be captured.

In a further step, the droplet 23 is captured by the activatedelectrodes 10′, depicted in FIG. 11 as liquid droplet 23 after delivery,wherein the liquid droplet 23 substantially covers the four activatedelectrodes 10′ (i.e. four capture electrodes). Despite the capturing ofthe liquid droplet 23, there is still sufficient room for the carrierfluid to pass by the liquid droplet 23 via remaining open part of theinlet port 19′, i.e. via the transfer zone 63.

The term “covering” describes a geometrical overlapping configuration ina projection longitudinally to an axis of the inlet port and/or along anaxis of flow exiting the opening area of the inlet port. Thiscorresponds to a visual appearance viewed along an optical axis, whichis perpendicular to the electrode array in a direction towards theelectrode array. In an actual configuration, further elements may or maynot be present between the opening of the inlet port 19′ and the one ormore electrodes 10′,10″ such as a hydrophobic foil, a processing liquid,an empty space in the gap or an electrowetting filler liquid.

FIG. 12 shows—in reference to FIG. 11—a schematic depiction of severalinlet port and capture electrode configurations:

a) 5-50% coverage;b) 50% coverage; andc) 55-95% coverage.

In a preferred configuration, the coverage is approximately 50%, otherconfigurations are also possible, e.g. between 5% and 95% of the openingarea of the inlet port, between 10% and 90% or between 25% and 75%.These configurations provide sufficient room for a transfer zone that isconfigured to provide a passage for a carrier liquid next to themicrofluidic droplet while processing liquid is captured in the capturezone.

FIG. 13 shows—in reference to FIG. 11—a schematic depiction amicrofluidic droplet being captured by multiple electrodes:

a) one electrode activated for accumulating and capturing processliquid, andb) four electrodes activated for accumulating and capturing biggeramounts of process liquid.

During the capturing of an initial droplet neighboring electrodes areactivated for enlarging the droplet and moving the already capturedliquid over a plurality of electrodes. The neighboring electrodes can befurther capturing electrodes or transport electrodes.

Preferred dimensions and materials are pointed to in table 1. Theseindications of materials and dimensions serve as preferred exampleswithout limiting the scope of the present invention.

TABLE 1 Part No Material Dimensions and Shape Droplet 23 aqueous Vo1ume:0.1-5 μl Substrate 11 PCB; — Synth. Polymer Electrodes 10 Al; Cu; Au; PtPlating: 1.5 × 1.5 mm Electrode Array  9 Electrodes 1 or 2 dimensional  9′ Film  3 Fluorinated thickness: 8-50 μm ethylene propylene (FEP),Cyclo olefin polymer (COP), Polypropylene (PP) Hydrophobic 17 Teflon ®(PTFE), thickness: 8-50 μm surface COP, FEP, PP, Coating: 2-200 nm CytopSpin coating: 5-500 nm, preferably 20 nm Rigid cover  4 Mylar ®;acrylic; 65 × 85 mm; Polypropylene (PP) Plate: 0.5-25.0 mm, preferably1.5 mm Gap  6 — 0.2-2.0 mm, preferably 0.5 mm Pipetting 19 — Diameter:0.3-3.0 mm orifice Spacer,  5 Polypropylene Frame: 0.2-2.0 mm, Gasket(PP), Synthetic preferably 0.5 mm or natural rubber Carrier liquid 60Silicon oil Volume: 1 μl-10 ml

REFERENCE SIGNS LIST  1 electrowetting sample processing system  2disposable cartridge  3 bottom layer   3′ membrane  3″ hydrophobic layer 4 top layer  5 spacer  6 gap between 3 and 4  7 base unit  8 cartridgeaccommodation site 9, 9′ electrode array 10 electrode  10′ activatedelectrode   10″ non-activated electrode 11 bottom substrate  11′ supportelement 12 cover plate 13 top substrate 14 central control unit 15electrically conductive material 16 hinge 17 hydrophobic surface 18piercing facility 19 through hole  19′ inlet port   19″ channel 20piercing pipette tip 21 compartment 22 additional piercing facility 23liquid droplet 24 dielectric layer 26 disposable pipette tip 27 piercingpin 40 board accommodation site 41 electrode board 42 electrical boardcontact elements 44 PCB border 50 supply channel 60 carrier liquid 61processing liquid 62 capture zone  62′ capture electrode 63 transferzone 70 droplet detector 71 droplet detector

1: A cartridge (2), in particular a disposable cartridge for use in anelectrowetting sample processing system, the cartridge (2) comprising atleast one inlet port (19′) for introducing an input liquid (60,61) in aninternal gap (6) of the cartridge (2), wherein the gap (6) comprises atleast one hydrophobic surface (17) and is configured to provide anelectrowetting induced movement of a microfluidic droplet (23) of inputliquid (60,61), wherein the input liquid (60,61) comprises a carrierliquid (60) and a processing liquid (61) and the gap (6) comprises acapture zone (62) that is configured to capture at least a part of theprocessing liquid (60,61) as a microfluidic droplet (23) by use ofelectrowetting force and the gap (6) further comprises a transfer zone(63) that is configured to provide a passage for the carrier liquid (60)next to the microfluidic droplet (23), while processing liquid (61) iscaptured in the capture zone (62). 2: The cartridge according to claim1, comprising a first part (4) with the inlet port (19′) and a secondpart (3) attached to the first part, such that the gap (6) is formedbetween the first part and the second part. 3: The cartridge accordingto claim 2, wherein the first part (4) comprises a rigid body and/or thesecond part (3) comprises or is an electrode support element (11′) or aflexible film (3′), in particular a polymer film and/or an electricallyisolating film. 4: The cartridge according to claim 2, wherein the gap(6) is defined by a spacer (5) that is arranged between the first partand the second part and/or by the shape of at least one of the two partsof the cartridge, in particular by a flexible part or a rigid part ofthe cartridge, and wherein in particular the second part is attached toa peripheral side structure of the first part. 5: The cartridgeaccording to claim 1, comprising at least one electrode (10), inparticular an electrode array (9), for applying an electrowetting forceto the microfluidic droplet (23). 6: The cartridge according to claim 1,comprises an inlet channel (19″) for transferring the processing liquidfrom the inlet port (19′) to the gap (6), wherein in particular theinlet channel (19″) is arranged substantially perpendicular or parallelto the orientation of the gap. 7: The cartridge according to claim 1,comprises an inlet channel (19″) for transferring the processing liquidfrom the inlet port (19′) to the gap (6), wherein in particular theinlet channel (19″) is arranged substantially perpendicular or parallelto the orientation of the gap. 8: The cartridge according to claim 1,configured to capture the processing liquid (61), which comprises atleast one of: a reagent liquid, a buffer, a diluent, an extractionliquid, a washing liquid and a suspension, which further in particularis a suspension of magnetic beads, single cells or cell aggregates. 9:The cartridge to claim 1, configured to be operated with a carrierliquid (60) that is a carrier liquid, in particular an electrowettingfiller liquid, further in particular a silicone oil. 10: The cartridgeaccording to claim 1, configured to receive the input liquid (60,61), inwhich the carrier liquid (60) sequentially and/or alternatingly enclosesthe processing liquid (61). 11: The cartridge according to claim 1,configured to provide the transfer zone (63) by an open space, which islocated between the inlet port (19′) and the top of the microfluidicdroplet (23) captured in the capture zone (62). 12: The cartridgeaccording to claim 1, comprising at least one capture zone (63) that islocated closest to the inlet port (19′) such that the area of thecapture zone covers between 5% and 95% of the opening area of the inletport (19′), in particular between 10% and 90%, further in particularbetween 25% and 75%. 13: The cartridge according to claim 1, comprisingat least one capture electrode that is located closest to the inlet port(19′) such that the area of the capture electrode covers between 5% and95% of the opening area of the inlet port (19′), in particular between10% and 90%, further in particular between 25% and 75%. 14: Thecartridge according to claim 1, configured to receive the processingliquid (61) that comprises multiple parts, in particular parts ofdifferent compositions, and to accumulate these parts for providing themicrofluidic droplet. 15: The cartridge according to claim 1, configuredto receive at least one part of the processing liquid (61) thatcomprises a volume that is insufficient for a transportation byelectrowetting and/or that comprises a volume of less than 2 μl, inparticular less than 1.5 μl. 16: The cartridge according to claim 1,configured to capture or to accumulate a microfluidic droplet (23) ofless than 10 μl in volume, in particular of less than 3 μl in volume.17: The cartridge according to claim 1, wherein the inlet port (19′)comprises a sealing surface (82) for a tube (51) to be inserted into theinlet port (19′), wherein in particular the inlet port (19′) isfunnel-shaped with an enlarged opening towards the tube (51) to beinserted. 18: An electrowetting sample processing system (1), inparticular a biological sample processing system comprising a cartridge(2) according to claim
 1. 19: An electrowetting sample processing system(1) comprising at least one inlet port (19′) for introducing an inputliquid and an internal gap (6) that comprises at least one hydrophobicsurface (17) and that is configured to manipulate a microfluidic droplet(23) separated from the input liquid (60,61), if an electrowetting forceis applied to the at least one microfluidic droplet, wherein the inputliquid comprises a processing liquid (61) and a carrier liquid (60) andthe gap comprises a capture zone (62) that is configured to capture atleast a part of the processing liquid by use of electrowetting force andthe gap further comprises a transfer zone (63) that is configured toprovide a passage for the carrier liquid from the inlet port to the gap,while processing liquid is captured in the capture zone. 20: Theelectrowetting sample processing system according to claim 18,comprising at least one electrode (10), in particular an electrode array(9), for applying an electrowetting force to the processing liquidand/or the microfluidic droplet (23). 21: The electrowetting sampleprocessing system according to claim 20, wherein at least one electrode(10) comprises at least one capture electrode (62′) that is configuredto capture at least a part of the processing liquid as a microfluidicdroplet by use of electrowetting force, wherein in particular the edgeof the capture electrode is arranged with an offset from the axis offlow of the inlet port (19′), further in particular with an offset of atleast a quarter or at least half of a largest diameter of the captureelectrode. 22: The electrowetting sample processing system according toclaim 21, wherein the one of the at least one capture electrode (62′)located closest to the inlet port (19′) covers between 5% and 95% of theopening area of the inlet port (19′), in particular between 10% and 90%,further in particular between 25% and 75%. 23: The electrowetting sampleprocessing system according to claim 20, wherein the at least oneelectrode (10) comprises a transport electrode (10′) for removing themicrofluidic droplet from the capture zone. 24: The electrowettingsample processing system according to claim 18, comprising a liquidfeeder (50) that is operatively connected to the inlet port (19′) by atube (51), in particular a flexible tube, for feeding an input liquid(60,61) of predetermined volume to the inlet port. 25: Theelectrowetting sample processing system according to claim 24, whereinthe liquid feeder (50) is configured to provide the input liquid (60,61)as at least one sequential and/or alternating feed of the processingliquid (61) and the carrier liquid (60). 26: The electrowetting sampleprocessing system according to claim 18, comprises a detector (70,71)for monitoring the feed of the input liquid (60,61), in particular theprocessing liquid (61) and/or the carrier liquid (60). 27: Theelectrowetting sample processing system according to claim 18, comprisesa controller for operating the liquid feeder (50), in particular adroplet generator, independently and/or asynchronously from theoperation of electrodes used for electrowetting. 28: A method foroperating the cartridge or the sample processing system according toclaim
 18. 29: A method for operating a cartridge (2) that comprises atleast one inlet port (19′) and an internal gap (6) with a capture zone(62) and a transfer zone (63), the method comprising: providing an inputliquid (60,61) that comprises a processing liquid (61) and a carrierliquid (60); separating at least a part of the processing liquid (61) inthe capture zone (62) by use of electrowetting force; transferring thecarrier liquid (60) from the inlet port (19′) to the gap (6) via thetransfer zone (63), while the processing liquid (61) is captured in thecapture zone (62); and capturing at least a part of the processingliquid (61) in the capture zone (62) for providing a microfluidicdroplet (23) that is movable by applying an electrowetting force to themicrofluidic droplet (23). 30: The method according to the claim 28,wherein the step of providing the input liquid (60,61) is accomplishedby sequentially and/or alternatingly feeding the processing liquid (61)and the carrier liquid (60). 31: The method according to claim 28,wherein the input liquid (60,61) comprises multiple liquid parts, inparticular parts of different compositions, and the capturing isaccomplished by accumulating these parts for providing the microfluidicdroplet (23). 32: The method according to claim 31, wherein the inputliquid (60,61) comprises at least one part that comprises a volume thatis insufficient for a transportation by electrowetting and/or thatcomprises a volume of less than 2 μl, in particular less than 1.5 μl.33: The method according to claim 28, comprising sequentially actuatingelectrodes for inducing a motion of the microfluidic droplets away fromthe capture zone (62), thereby enabling a following part of theprocessing liquid (61) to be captured.